Production of new animal models to determine how CTLA-4 contributes to T1D susceptibility. This application addresses Challenge Area 15: Translational Sciences and specific Challenge Topic 15-DK-102: Develop improved animal models of NIDDK diseases. The genetic basis of T1D has been intensely investigated. More than 20 and 5 T1D-susceptibility genes have been mapped in rodents and humans respectively, with MHC polymorphisms providing the best predictive measure of risk in both species. Ctla4 is the only other T1D susceptibility gene common to humans and mice. It encodes for a protein belonging to the T cell costimulatory family whose expression is induced upon T cell activation and which functions to inhibit T cell activation. CTLA-4 is also expressed constitutively on the regulatory FOXP3+ CD4+ T (Treg) cell subset. Treg cells are critical for preventing T1D in mice and a functional link between CTLA-4 and Treg cells in controlling T1D has been suggested, especially given that mice lacking CTLA-4 or FOXP3 succumb to multi-organ failures caused by unchecked self-reactive T cell expansion and infiltration into tissues leading to death before 4 weeks of age. Recently, we and others have definitively shown that CTLA-4 is essential for normal Treg cell function using new animal models with T cell subset-restricted expression of CTLA-4. With this insight, and a realigned research focus on where CTLA-4 functions, the goal of this application is to generate more rationally designed animal models to understand how CTLA-4, in conjunction with other disease susceptibility parameters, impact T1D progression. When, and to what extent, aberrations in CTLA-4 function can specifically lead to T cell mediated destruction of [unreadable]-islet cells are unresolved issues. The difficulty in generating animal models to dissect CTLA-4 function has been a major impediment to research progress. It has been particularly challenging to study CTLA-4 function separately in conventional T cells and Treg cells in vivo, since manipulations of the latter subset result in profound deregulation of T cell homeostasis and early mortality. As a result, there are no well-defined animal models of T1D progression that involves alterations in CTLA-4 function. By simply restricting CTLA-4 expression to particular T cell subsets, we have now generated a mouse model of T1D in diabetes resistant C57BL/6 (B6) background, which develops spontaneous insulitis with 100% penetrance, but does not progress to full-blown diabetes. These mice, referred to as CT4act, are one of a series of mouse models that target CTLA-4 expression to distinct T cell subsets. Using these models, we have shown that Treg cells maintain T cell homeostasis in a CTLA-4-dependent manner. CT4act mice, where CTLA-4 is expressed only in activated T cells, are afflicted with lymphoproliferation due to functionally impaired Treg cells that lack CTLA-4. However, they live to 8-12 months of age instead of 3-4 weeks for Ctla4-/- mice. This relative longevity can be attributed to a paucity of activated T cells in non-lymphoid tissues in CT4act mice, suggesting that CTLA-4 is required in aberrantly activated T cells to prevent the infiltration of pathogenic T cells into non-lymphoid tissues. One exception to this protection afforded by CTLA-4 for tissue integrity is the [unreadable]-islet cells of the pancreas. These results strongly suggest that Treg cells prevent insulitis in a CTLA-4-dependent manner. Using CT4act mice as the main platform, we are generating a series of B6 mouse models of T1D that can fully recapitulate disease progression to determine what processes control insulitis to diabetes transition. Towards this purpose, we have already generated several mouse models that will be used to assess the relative contributions of CTLA-4-independent function of Treg cells in conjunction with MHC alleles, and TCR repertoire for pancreatic self-antigens on Treg and pathogenic T cells. Once the critical parameter controlling diabetes induction is established, we will utilize mouse models in which CTLA-4 can be ablated in a temporally-regulated and cell type-specific manner to determine how CTLA-4 impacts T1D checkpoint parameters. PUBLIC HEALTH RELEVANCE: Impaired CTLA-4 function has been strongly implicated as a major factor in T1D susceptibility in humans and rodents. This application will generate new mouse models to determine the relationship between CTLA-4 and other factors that regulate T1D progression so that more rationale therapies designed to target CTLA-4 to treat T1D can be formulated.